Lyme disease is a progressive, systemic infection caused by the spirochete Borrelia burgdorferi. The disease is transmitted to man by the bite of the deer tick (Ixodes scapularis and other species). Diagnostic tests for Lyme disease rely mainly on the detection of human antibodies to spirochetal antigens. The principal test used for screening human sera for antibodies to the Lyme spirochete is enzyme-linked immunosorbent assay (ELISA). Due to the significant inaccuracies inherent in ELISA, sera which are ELISA-positive or indeterminate are often subjected to a confirmatory test. The confirmatory test now in most common use and officially recommended by the U.S. Centers for Disease Control (CDC) is the Western blot. In a conventional Western blot assay, antigens of a given pathogen are resolved into discrete bands on the surface of a paper-like nitrocellulose membrane. The serum to be tested is allowed to react with the antigen bands, and serum antibodies which bind specific bands are detected with a labeled anti-human antibody reagent. Typically, results of the Western blot test appear as a series of bands on a membrane strip. The pattern of bands is compared with the band pattern of known positive sera to produce a diagnostic result. The exact position of bands, and the number of bands which correlate with positivity, differ depending on the pathogen.
Considerable effort has been expended to develop new and improved diagnostic tests for Lyme disease. For example, U.S. Pat. No. 5,187,065 discloses methods of detecting Lyme disease in mammals that otherwise show seronegativity due to the generation of immune complexes which "hide" the antibodies raised to the spirochete; disassociation of such complexes followed by immunological assay procedures such as ELISA are described. U.S. Pat. No. 5,470,712 provides bioassays which incorporate non-flaggellar B. burgdorferi proteins, or antibodies raised to such proteins, to create an assay where such proteins or antibodies are bound to a surface and form complexes with certain components of the serum. Similarly, U.S. Pat. No. 5,308,753 teaches the formation of assays which may be used to diagnose Lyme and other diseases which induce primary or secondary IgM antibody-mediated immunity. U.S. Pat. No. 5,217,872 teaches a method of detecting B. burgdorferi antigens through an assay which utilizes vesicle proteins released from the spirochete, while U.S. Pat. Nos. 5,494,797 and 5,324,630 teach the detection of the Lyme spirochete via oligonucleotide probes. U.S. Pat. No. 4,888,276 describes a reliable, noninvasive method for detecting antigens of B. burgdorferi from the urine of affected individuals and U.S. Pat. No. 5,155,022 teaches an improved method of assaying for Lyme disease by eliminating crossreacting antibodies. Both U.S. Pat. Nos. 4,859,419 and 5,100,626 provide apparati that are able to assay multiple samples for a specific disease such as Lyme. However, none of these patents teach a method of simultaneously assaying for Lyme disease and other diseases.
The same ticks which transmit Lyme disease to humans also transmit babesiosis, an underdiagnosed parasitic infection which may have serious consequences. In the United States, the major pathogen is the protozoan Babesia microti; in Europe and other countries, other species of Babesia including B. divergens, B. bovis and B. bigemina, all known pathogens of cattle, have been implicated. Despommier, D. et al., Parasitic Diseases, Springer-Verlag, New York (1995). Ehrlichiosis, caused by either of two rickettsial Ehrlichia species, is another disease which the Ixodid tick may transmit. In addition, a babesiosis-like illness in the northwestern United States has been attributed to an unidentified Babesia-like organism, thus far termed WA1. Quick, R. et al., Annals of Int. Med. 119: 284-290 (1993).
The clinical symptoms and severity of babesiosis cover a wide spectrum. While the disease is subclinical in most cases, it may be severe to fatal in others. Telford, S. R. III et al., Topley and Wilson's Microbiology, in press; Herwaldt, B. L. et al., Am. J. Trop. Med. Hyg. 53(2): 146-151; Garcia, L. S. et al., Diagnostic Medical Parasitology, Washington, D.C., p. 131-135 (Second Edition, 1993); Boustani, M. R. et al., Am. J. Respir. Crit. Care. Med. 149: 1689-1691 (1994); Horowitz, M. et al., Chest 106(4): 1299-1301 (1994); Rosenbaum, G. S. et al., Clin. Infect. Dis. 20: 203-204 (1995); Machtinger, L. et al., J. Clin. Apheresis 8: 78-81 (1993). Splenectomy, immunosuppression, and advanced age are significant prognostic indicators. Early symptoms may appear within one to several weeks post-infection, and typically include malaise, anorexia, and fatigue. In susceptible individuals, these progress quickly to more serious symptoms, including fever up to 40.degree. C., sweating, myalgia, nausea, vomiting, headache, shaking chills, emotional lability and depression, hemoglobinuria, hyperesthesia, and pulmonary edema. Blood analyses may reveal anemia, thrombocytopenia, and low white blood cell count, while lactic dehydrogenase, bilirubin and transaminases may appear at elevated levels. The wide range in symptoms makes the clinical diagnosis of babesiosis difficult, and additionally so in view of the possibility of either confusion or coinfection with Lyme disease and/or ehrlichiosis.
Co-infected patients may be subject to more severe illness than caused by either pathogen alone. Significant frequencies of co-infection have been reported in areas endemic for babesiosis and Lyme disease. Mitchell, P. D. et al., J. Clin. Microbiol. 34: 724-727 (1996). Both diseases appear to be rising in incidence, perhaps due to changes both in public awareness of tick-bome diseases and in the interactions of man and the surrounding natural environment. Because therapeutic treatment and prognosis differ for the various diseases, accurate diagnosis is essential for successful clinical management of the patient.
Babesia infects and multiplies within the erythrocytes of the host and thus laboratory testing for babesiosis has traditionally been based on examination of Giemsa-stained blood smears. Telford, S. R. III et al., Topley and Wilson's Microbiology, in press; Diagnostic Medical Parasitology, Washington, D.C., p. 131-135 (Second Edition, 1993). Babesia may be visualized in parasitized erythrocytes as pear-shaped piroplasms (hence the common name for babesiosis, "piroplasmosis") or rings, and infrequently as tetrads (maltese cross forms) which are considered as definitive evidence of infection. Parasitemia in infected individuals may vary between 1-20%, while in splenectomized patients, it may attain 85%. Nevertheless, low level parasitemia is common enough so that failure to observe the parasite in blood smears does not prove the absence of infection with Babesia. Babesia may also be revealed by inoculation of patient blood samples into hamsters, which develop high levels of parasitemia; however, this technique may require up to 6 weeks to yield detectable results.
Serology provides a useful diagnostic approach for babesiosis. Telford, S. R. III et al., Topley and Wilson's Microbiology, in press; Diagnostic Medical Parasitology, Washington, D.C., p. 131-135 (Second Edition, 1993). IgM and IgG antibodies to Babesia are produced by infected individuals, and may be detected where direct visual evidence of parasitemia is lacking. An indirect immunofluorescence assay has been developed and has been applied as a diagnostic method. Krause, P. J. et al., J Infect. Dis. 169: 923-926 (1994); Chisholm, E. S. et al., Am. J. Trop. Med. Hyg. 27: 14-19 (1978); Chisholm, E. S. et al., Am. J. Trop. Med. Hyg. 35: 921-925 (1986). Immunofluorescence testing of babesiosis patient sera has shown very little crossreactivity between B. microti and the WA1 stain found in some Washington State patients. Telford, S. R. III et al., Topley and Wilson's Microbiology, in press; Quick, R. E. et al., Ann Intem. Med. 119(4): 284-290 (1993).
In another diagnostic method, Babesia DNA is detected by polymerase chain reaction (PCR). Persing, S. et al., BioTechniques 17: 788-791 (1994). While the sensitivity of PCR is one of its main advantages, PCR at present remains a technique beyond the expertise of the average diagnostic laboratory. In inexperienced hands, PCR may lead to an inaccurate diagnosis. Thus, while it would be preferable to utilize a rapid and accurate screening method for diagnosing Babesiosis, especially one which could be run simultaneously with tests for Lyme and other diseases, none have thus been developed.
It would thus be desirable to provide a method for detecting Lyme disease as well as at least one additional disease. It would further be desirable that such a method combine the confirmatory Western blot detection of Lyme disease with a screening test for diseases which may accompany Lyme disease during its transmission. It would also be desirable that such a method be able to screen for diseases such as babesiosis and ehrlichiosis. It would further be desirable that such a method have the capability of being automated. It would additionally be desirable to provide a method wherein initial screening is followed with a confirmatory test. It would further be desirable to provide a method wherein the confirmatory test is a Western blot assay.